Mass spectrometry assay for estrogenic compounds

ABSTRACT

Methods are provided for detecting the amount of one or more HRT panel analytes (i.e., estrone (E1), estrone sulfate (E1s), 17α-estradiol (E2a), 17β-estradiol (E2b), estradiol sulfate (E2s), estriol (E3), equilin (EQ), 17α-dihydroequilin (EQa), 17β-dihydroequilin (EQb), Equilenin (EN), 17α-dihydroequilenin (ENa), 17β-dihydroequilenin (ENb), and Δ8,9-dehydroestrone (dE1)) in a sample by mass spectrometry. The methods generally involve ionizing one or more HRT panel analytes in a sample and quantifying the generated ions to determine the amount of one or more HRT panel analytes in the sample. In methods where amounts of multiple HRT panel analytes are detected, the amounts of multiple analytes are detected in the same sample injection.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/641,227, filed Dec. 17, 2009, which claims priority to U.S. Appl.No. 61/161,160, filed Mar. 18, 2009, and U.S. Appl. No. 61/140,575,filed Dec. 23, 2008, each of which is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

The invention relates to methods for measurement of certain estrogeniccompounds, in particular by tandem mass spectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Hormone replacement therapy (HRT) is a treatment to artificially boostthe hormone levels for surgically menopausal, perimenopausal andpostmenopausal women. The main hormones involved in HRT are estrogens,progestins and sometimes testosterone. They can be delivered to the bodyvia patches, tablets, creams, gels, troches, IUDs, and injections(rarely) in dosages of sequentially combined HRT (scHRT) or continuouscombined HRT (ccHRT). HRT is also useful for treatment of menopausalsymptoms.

The most commonly and widely prescribed form of HRT is Premarin®.Premarin® is a compound drug consisting of 10 biologically activeestrogens or conjugated equine estrogens (estrone (E1), equilin (EQ),17α-dihydroequilin (EQa), 17α-estradiol (E2a), 17β-dihydroequilin (EQb),17α-dihydroequilenin (ENa), 17β-dihydroequilenin (ENb), equilenin (EN),17β-estradiol (E2b), Δ8,9-dehydroestrone (dE1), and conjugates thereof).These estrogens are present mainly as “conjugates,” i.e., modifiedchemical forms in which the active estrogen is coupled to anotherchemical group such as sulfate. After being taken into the woman's body,the conjugated estrogens of Premarin® are converted to the activeunconjugated estrogens or excreted from the woman's body. A number ofclinical trials have confirmed the effectiveness of HRT in preservingand increasing bone mineral density in postmenopausal women.

However, a link between estrogens and various increased health risks,such as developing breast cancer or various cardiovascular conditions,has been reported. See The Women's Health Initiative Steering Committee,JAMA 2004, 14:1701-12; and The Writing Group for the Women's HealthInitiative Investigators, JAMA 2002, 3:321-333.

Previously, methods have been reported for measuring various estrogensaffected by HRT. For example, Goldman, et al., U.S. patent applicationSer. Nos. 11/946, 017, 12/002,314, and 12/328,735, describe measurementof estradiol and estrone by mass spectrometry techniques; Chen, H. C.,et al., Chemosphere 2008, Epub ahead of print; and Alavarez, S., et al.,J. Chromatogr A 2008, 1201:46-54 report measuring estrone, estradiol,and estriol using liquid chromatography tandem mass spectrometry inwastewater samples and urine, respectively. Qin, F., et al., Anal Chem.2008, 80:3404-11, reported hydrophilic interaction liquid chromatographywith tandem mass spectrometry for detection of urinary estrogenconjugates. Reepmeyer, J. C., et al., J. Chromatogr A 2005, 1083:42-51,reported detection of equilin-3-sulfate andΔ8,9-dehydroestrone-3-sulfate by liquid chromatography tandem massspectrometry using carbon-coated zirconia and porous graphitic carbonstationary phases.

SUMMARY OF THE INVENTION

Methods are provided for detecting the amount of one or more (andpreferably multiple) HRT panel analytes (i.e., estrone (E1), estronesulfate (E1s), 17α-estradiol (E2a), 17β-estradiol (E2b), estradiolsulfate (E2s), estriol (E3), equilin (EQ), 17α-dihydroequilin (EQa),17β-dihydroequilin (EQb), Equilenin (EN), 17α-dihydroequilenin (ENa),17β-dihydroequilenin (ENb), and Δ8,9-dehydroestrone (dE1)) in a sampleby mass spectrometry, including tandem mass spectrometry. In methodswhere amounts of multiple HRT panel analytes are detected, the amountsof multiple analytes are detected in the same sample injection.

These methods include: subjecting a sample, purified by methodsdescribed below, to ionization under conditions suitable to produce oneor more ions detectable by mass spectrometry from each of one or moreHRT panel analytes; determining the amounts of one or more ions fromeach of the one or more HRT panel analytes by tandem mass spectrometry;and using the amounts of the one or more ions from each of the one ormore HRT panel analytes to determine the amounts of each of the one ormore HRT panel analytes in the sample. Preferably, the sample is abiological fluid; more preferably the sample is serum.

Some embodiments presented herein may be useful for determining thelevel of two or more circulating estrogenic compounds in an individualundergoing hormone replacement therapy (HRT). In related embodiments, aratio may be calculated between the levels of two or more circulatingestrogenic compounds in an individual. In these embodiments, the ratiois a comparison of the combined levels of one or more sulfatedestrogenic compounds and the combined levels of one or more non-sulfatedestrogenic compounds. In these embodiments, the predominance of thenon-sulfated form may indicate that a decrease in the individual's HRTdosage may be desirable. Alternatively, if the sulfated form ispredominant, an increase in the individual's HRT dosage may bedesirable. In certain preferred embodiments, the non-sulfated estrogeniccompound(s) comprise the non-sulfated versions of the sulfatedestrogenic compound(s). In these embodiments, the one or more sulfatedestrogenic compounds comprise one or more estrogenic compounds selectedfrom the group consisting of estrone sulfate (E1s) and estradiol sulfate(E2s).

In preferred embodiments, the amounts of two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, ten or more, eleven or more, twelve or more, or thirteenor more HRT panel analytes are determined.

In embodiments where only one HRT panel analyte or estrogenic compoundis determined, the HRT panel analyte or estrogenic compound excludes E1.In some embodiments where only two HRT panel analytes or estrogeniccompounds are determined, the two HRT panel analytes or estrogeniccompounds exclude the combination of E1 and E3 or the combination of E1Sand E2S. In some embodiments where only three HRT panel analytes orestrogenic compounds are determined, the three HRT panel analytes orestrogenic compounds exclude the combination of E1, E2b and E3. Inembodiments where the sample is subjected to turbulent flow liquidchromatography prior to ionization, the specific combinations indicatedabove of two or three HRT panel analytes or estrogenic compounds may notbe excluded. All of the above embodiments apply to detection ofestrogenic compounds in an sample including where the sample is takenfrom an individual undergoing hormone replacement therapy.

In some embodiments, one of the one or more HRT panel analytes is E1. Inrelated embodiments, one or more ions from E1 comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 269.10±0.50, 145.10±0.50, and 143.09±0.50. In particularlypreferred embodiments, one or more ions from E1 comprise a precursor ionwith m/z of 269.10±0.50, and one or more fragment ions selected from thegroup of ions with m/z of 145.10±0.50 and 143.09±0.50.

In some embodiments, one of the one or more HRT panel analytes is E1s.In related embodiments, one or more ions from E1s comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 349.01±0.50, 145.10±0.50, and 143.08±0.50. In particularlypreferred embodiments, one or more ions from E1s comprise a precursorion with m/z of 349.01±0.50, and one or more fragment ions selected fromthe group of ions with m/z of 145.10±0.50 and 143.08±0.50.

In some embodiments, one of the one or more HRT panel analytes is E2a.In related embodiments, one or more ions from E2a comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 271.12±0.50, 145.10±0.50, and 143.1±0.50. In particularlypreferred embodiments, one or more ions from E2a comprise a precursorion with m/z of 271.12±0.50, and one or more fragment ions selected fromthe group of ions with m/z of 145.10±0.50 and 143.1±0.50.

In some embodiments, one of the one or more HRT panel analytes is E2b.In related embodiments, one or more ions from E2b comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 271.12±0.50, 183.10±0.50, and 169.10. In particularly preferredembodiments, one or more ions from E2b comprise a precursor ion with m/zof 271.12±0.50, and one or more fragment ions selected from the group ofions with m/z of 183.10±0.50 and 169.10.

In some embodiments, one of the one or more HRT panel analytes is E2s.In related embodiments, one or more ions from E2s comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 351.02±0.50, 183.10±0.50, and 145.10±0.50. In particularlypreferred embodiments, one or more ions from E2s comprise a precursorion with m/z of 351.02±0.50, and one or more fragment ions selected fromthe group of ions with m/z of 183.10±0.50 and 145.10±0.50.

In some embodiments, one of the one or more HRT panel analytes is E3. Inrelated embodiments, one or more ions from E3 comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 287.10±0.50, 171.10±0.50, and 143.10±0.50. In particularlypreferred embodiments, one or more ions from E3 comprise a precursor ionwith m/z of 287.10±0.50, and one or more fragment ions selected from thegroup of ions with m/z of 171.10±0.50 and 143.10±0.50.

In some embodiments, one of the one or more HRT panel analytes is EQ. Inrelated embodiments, one or more ions from EQ comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 267.06±0.50, 181.07±0.50, and 115.07±0.50. In particularlypreferred embodiments, one or more ions from EQ comprise a precursor ionwith m/z of 267.06±0.50, and one or more fragment ions selected from thegroup of ions with m/z of 181.07±0.50 and 115.07±0.50.

In some embodiments, one of the one or more HRT panel analytes is EQa.In related embodiments, one or more ions from EQa comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 269.10±0.50, 183.11±0.50, and 169.10±0.50. In particularlypreferred embodiments, one or more ions from EQa comprise a precursorion with m/z of 269.10±0.50, and one or more fragment ions selected fromthe group of ions with m/z of 183.11±0.50 and 169.10±0.50.

In some embodiments, one of the one or more HRT panel analytes is EQb.In related embodiments, one or more ions from EQb comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 269.09±0.50, 193.10±0.50, and 143.10±0.50. In particularlypreferred embodiments, one or more ions from EQb comprise a precursorion with m/z of 269.09±0.50, and one or more fragment ions selected fromthe group of ions with m/z of 193.10±0.50 and 143.10±0.50.

In some embodiments, one of the one or more HRT panel analytes is EN. Inrelated embodiments, one or more ions from EN comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 265.09±0.50, 221.08±0.50, and 193.10±0.50. In particularlypreferred embodiments, one or more ions from EN comprise a precursor ionwith m/z of 265.09±0.50, and one or more fragment ions selected from thegroup of ions with m/z of 221.08±0.50 and 193.10±0.50.

In some embodiments, one of the one or more HRT panel analytes is ENa.In related embodiments, one or more ions from ENa comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 267.08±0.50, 195.13±0.50, and 180.10±0.50. In particularlypreferred embodiments, one or more ions from ENa comprise a precursorion with m/z of 267.08±0.50, and one or more fragment ions selected fromthe group of ions with m/z of 195.13±0.50 and 180.10±0.50.

In some embodiments, one of the one or more HRT panel analytes is ENb.In related embodiments, one or more ions from ENb comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 267.08±0.50, 195.12±0.50, and 180.09±0.50. In particularlypreferred embodiments, one or more ions from ENb comprise a precursorion with m/z of 267.08±0.50, and one or more fragment ions are selectedfrom the group of ions with m/z of 195.12±0.50 and 180.09±0.50.

In some embodiments, one of the one or more HRT panel analytes is dE1.In related embodiments, one or more ions from dE1 comprise ions selectedfrom the group consisting of negative ions with a mass to charge ratio(m/z) of 267.06±0.50, 195.08±0.50, and 171.07±0.50. In particularlypreferred embodiments, one or more ions from dE1 comprise a precursorion with m/z of 267.06±0.50, and one or more fragment ions selected fromthe group of ions with m/z of 195.08±0.50 and 171.07±0.50.

Embodiments of the present invention may involve the combination ofliquid chromatography with mass spectrometry. In some embodiments, theliquid chromatography may comprise HPLC, UPLC, TFLC, or any combinationthereof. For example, in some embodiments, HPLC, alone or in combinationwith one or more purification methods such as for example SPE (e.g.,TFLC) and/or protein precipitation and filtration, is utilized to purifyan analyte in a sample. In other embodiments, the liquid chromatographymay comprise UPLC, either alone or in combination with one or moreadditional purification methods, such as SPE (e.g., TFLC) and/or proteinprecipitation and filtration, to purify an analyte in a sample.

As used herein, hormone replacement therapy (HRT) is a treatment toartificially boost the reproductive hormone levels in women undergoingtreatment. The main reproductive hormones involved in HRT are estrogens,progestins and sometimes testosterone. They can be delivered to the bodyvia patches, tablets, creams, gels, troches, IUDs, and injections(rarely) in dosages of sequentially combined HRT (scHRT) or continuouscombined HRT (ccHRT).

In some embodiments, at least one purification step and massspectrometric analysis is conducted in an on-line fashion. In anotherpreferred embodiment, the mass spectrometry is tandem mass spectrometry(MS/MS).

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in negative ion mode. Alternatively, massspectrometry is performed in positive ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain preferred embodiments, one or moreestrogenic compounds and/or internal standards are ionized using heatedESI in negative ion mode.

In preferred embodiments, one or more separately detectable internalstandards is provided in the sample, the amount of which is alsodetermined in the sample. In these embodiments, all or a portion of oneor more endogenous analytes selected from the group consisting of HRTpanel analytes, and the one or more internal standards present in thesample are ionized to produce a plurality of ions detectable in a massspectrometer. In preferred embodiments, the amount of ions generatedfrom an analyte of interest may be related to the presence of amount ofanalyte of interest in the sample by comparison to one or more internalstandards.

Preferred internal standards include d₄-estrone (E1-d4),d₂-17α-estradiol (E2a-d2), d₅-17β-estradiol (E2b-d5), d₃-estriol(E3-d3), d₄-equilin (EQ-d4), d₅-17β-dihydroequilin (EQb-d5),d₃-equilenin (EN-d3), d₃-17β-dihydroequilenin (ENb-d3), d₄-estronesulfate (E1s-d4), d₄-17β-estradiol sulfate (E2bs-d4), andd₂-Δ8,9-dehydroestrone (dE1-d2). However, this listing of preferredinternal standards is not intended to be exclusive, i.e., other suitableinternal standards may be used.

In preferred embodiments, E1-d4 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 273.13±0.50, and 147.08±0.50. In particularlypreferred embodiments, E1-d4 ions comprise a precursor ion with m/z of273.13±0.50, and a fragment ion with m/z of 147.08±0.50.

In preferred embodiments, E2a-d2 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 273.20±0.50, and 147.08±0.50. In particularlypreferred embodiments, E2a-d2 ions comprise a precursor ion with m/z of273.20±0.50, and a fragment ion with m/z of 147.08±0.50.

In preferred embodiments, E2b-d5 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 276.13±0.50, and 187.08±0.50. In particularlypreferred embodiments, E2b-d5 ions comprise a precursor ion with m/z of276.13±0.50, and a fragment ion with m/z of 187.08±0.50.

In preferred embodiments, E3-d3 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 290.20±0.50, and 173.10±0.50. In particularlypreferred embodiments, E3-d3 ions comprise a precursor ion with m/z of290.20±0.50, and a fragment ion with m/z of 173.10±0.50.

In preferred embodiments, EQ-d4 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 271.16±0.50, and 243.14±0.50. In particularlypreferred embodiments, EQ-d4 ions comprise a precursor ion with m/z of271.16±0.50, and a fragment ion with m/z of 243.14±0.50.

In preferred embodiments, EQb-d5 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 274.15±0.50, and 213.10±0.50. In particularlypreferred embodiments, EQb-d5 ions comprise a precursor ion with m/z of274.15±0.50, and a fragment ion with m/z of 213.10±0.50.

In preferred embodiments, EN-d3 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 268.15±0.50, and 182.08±0.50. In particularlypreferred embodiments, EN-d3 ions comprise a precursor ion with m/z of268.15±0.50, and a fragment ion with m/z of 182.08±0.50.

In preferred embodiments, ENb-d3 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 270.17±0.50, and 182.08±0.50. In particularlypreferred embodiments, ENb-d3 ions comprise a precursor ion with m/z of270.17±0.50, and a fragment ion with m/z of 182.08±0.50.

In preferred embodiments, E1s-d4 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 352.9±0.50, and 147.09±0.50. In particularlypreferred embodiments, E1s-d4 ions comprise a precursor ion with m/z of352.9±0.50, and a fragment ion with m/z of 147.09±0.50.

In preferred embodiments, E2bs-d4 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 355.0±0.50, and 275.18±0.50. In particularlypreferred embodiments, E2bs-d4 ions comprise a precursor ion with m/z of355.0±0.50, and a fragment ion with m/z of 275.18±0.50.

In preferred embodiments, dE1-d2 ions detectable in a mass spectrometerare selected from the group consisting of positive ions with a mass tocharge ratio (m/z) of 269.02±0.50, and 209.05±0.50. In particularlypreferred embodiments, dE1-d2 ions comprise a precursor ion with m/z of269.02±0.50, and a fragment ion with m/z of 209.05±0.50.

In other embodiments, the amount of an analyte in a sample may bedetermined by comparison of the amount of one or more analyte ionsdetected by mass spectrometry to the amount of one or more standard ionsdetected by mass spectrometry in an external reference standard.Exemplary external reference standards may comprise blank plasma orserum spiked with a known amount of one or more of the above namedinternal standards and/or analytes of interest.

The features of the embodiments listed above may be combined withoutlimitation for use in methods of the present invention.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium,¹³C, and ¹⁵N. Deuterium is a useful label because it can potentiallyproduce three mass unit shifts in a labeled methylation product relativeto an unlabeled methylation product. For example, d₄-estrone has a massfour units higher than estrone. The iosotopic label can be incorporatedat one or more positions in the molecule and one or more kinds ofisotopic labels can be used on the same isotopically labeled molecule.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “sample” refers to any sample that may containan analyte of interest. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. For example,“body fluid” may include blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like. Preferred samples for use in thepresent invention comprise human serum and human plasma. Such samplesderived from an individual undergoing hormone replacement therapy,particularly serum or plasma, are useful for determining the level ofcirculating estrogenic compounds in the individual.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (also sometimes known as “high pressure liquid chromatography”)refers to liquid chromatography in which the degree of separation isincreased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column. As used herein, theterm “ultra high performance liquid chromatography” or “UPLC” or “UHPLC”(sometimes known as “ultra high pressure liquid chromatography”) refersto HPLC which occurs at much higher pressures than traditional HPLCtechniques.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow.” When fluid flowsslowly and smoothly, the flow is called “laminar flow.” For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns,” which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In somepreferred embodiments, the analytical column contains particles betweenabout 1.5 μm and 3.5 μm in diameter.

As used herein, the term “on-line” or “inline,” for example as used in“on-line automated fashion” or “on-line extraction,” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “sample injection” refers to introducing analiquot of a single sample into an analytical instrument, for example amass spectrometer. This introduction may occur directly or indirectly.An indirect sample injection may be accomplished, for example, byinjecting an aliquot of a sample into a HPLC or UPLC analytical columnthat is connected to a mass spectrometer in an on-line fashion.

As used herein, the term “same sample injection” with respect tomultiple analyte analysis by mass spectrometry means that the ions fortwo or more different analytes are determined essentially simultaneouslyby measuring ions for the different analytes from the same (i.e.identical) sample injection.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser desorption thermal desorption is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatscauses the sample to transfer into the gas phase. The gas phase sampleis then drawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of Total Ion Count for a single sample injectioncontaining thirteen HRT panel analytes. Details are discussed in Example4.

FIG. 2 shows HRT panel analyte peaks generated by TFLC followed byHPLC-MS/MS. Details are described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring the amount of one or more HRT panelanalytes in a sample. More specifically, mass spectrometric methods aredescribed for quantifying one or more HRT panel analytes in a samplethat typically has been purified by one or more steps prior to massspectrometry. The methods may utilize a liquid chromatography step suchas turbulent flow liquid chromatography (TFLC) to perform a purificationof selected analytes combined with methods of mass spectrometry (MS)thereby providing a high-throughput assay system for quantifying one ormore HRT panel analytes in a sample. The preferred embodiments areparticularly well suited for application in large clinical laboratoriesfor automated HRT monitoring.

Suitable samples for use in methods of the present invention include anysample that may contain one or more of the analytes of interest. In somepreferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as urine, blood, plasma, serum, saliva, and cerebrospinalfluid, or tissue samples; preferably plasma or serum; most preferablyserum. Such samples may be obtained, for example, from a patient; thatis, a living person, male or female, presenting oneself in a clinicalsetting for diagnosis, prognosis, or treatment of a disease orcondition. The sample is preferably obtained from a patient, forexample, blood serum.

When evaluating the result of analysis of a patient sample, a ratio maybe calculated comparing the amount of one or more sulfated analyte toone or more un-sulfated analyte. In preferred embodiments, the sulfatedand un-sulfated analytes comprise sulfated and un-sulfated forms of thesame analyte (for example, estrone sulfate:estrone). However, the numbertype of sulfated analytes utilized need not be the same as the numberand type of un-sulfated analytes utilized when determining the ratio.

A predominance of non-sulfated analytes may indicate that a decrease inthe individual's HRT dosage may be desirable. Alternatively, if sulfatedanalytes are predominant, an increase in the individual's HRT dosage maybe desirable.

The present invention also contemplates kits for a HRT monitoring assay.A kit for a HRT monitoring assay may include a kit comprising thecompositions provided herein. For example, a kit may include packagingmaterial and measured amounts of one or more isotopically labeledinternal standard, in amounts sufficient for at least one assay.Typically, the kits will also include instructions recorded in atangible form (e.g., contained on paper or an electronic medium) forusing the packaged reagents for use in a HRT monitoring assay.

Sample Preparation for Mass Spectrometry

Some or all HRT panel analytes in a sample may be bound to proteins,such as sex hormone binding globulin (SHBG) or albumin, if present inthe sample. Various methods may be used to disrupt the interactionbetween HRT panel analytes and protein prior to the implementation ofone or more enrichment steps and/or MS analysis so that the amount of aHRT panel analyte measured by mass spectrometry is a reflection of thetotal for that HRT panel analyte in the sample (e.g., free estradiol andestradiol bound to protein). Once HRT panel analytes and proteins havebeen separated in the sample, HRT panel analytes may be enrichedrelative to one or more other components in the sample (e.g. protein) byvarious methods known in the art, such as for example, liquidchromatography, filtration, centrifugation, thin layer chromatography(TLC), electrophoresis including capillary electrophoresis, affinityseparations including immunoaffinity separations, extraction methodsincluding ethyl acetate or methanol extraction, and the use ofchaotropic agents or any combination of the above or the like.

Protein precipitation is one preferred method of preparing a sample,especially a biological sample, such as serum or plasma. Such proteinpurification methods are well known in the art, for example, Polson etal., Journal of Chromatography B 785:263-275 (2003), describes proteinprecipitation techniques suitable for use in the methods. Proteinprecipitation may be used to remove most of the protein from the sampleleaving HRT panel analytes in the supernatant. The samples may becentrifuged to separate the liquid supernatant from the precipitatedproteins. The resultant supernatant may then be applied to liquidchromatography and subsequent mass spectrometry analysis. In certainembodiments, the use of protein precipitation such as for example,acetonitrile protein precipitation, obviates the need for turbulent flowliquid chromatography (TFLC) or other on-line extraction prior to HPLCand mass spectrometry. Accordingly in such embodiments, the methodinvolves (1) performing a protein precipitation of the sample ofinterest; and (2) loading the supernatant directly onto the HPLC-massspectrometer without using on-line extraction or turbulent flow liquidchromatography (TFLC).

In other preferred embodiments, HRT panel analytes may be released froma protein without having to precipitate the protein. For example, anaqueous formic acid solution may be added to the sample to disruptinteraction between a protein and a HRT panel analyte. Alternatively,ammonium sulfate may be added to the sample to disrupt ionicinteractions between a carrier protein and a HRT panel analyte withoutprecipitating the carrier protein.

In some preferred embodiments, TFLC, alone or in combination with one ormore purification methods, may be used to purify HRT panel analytesprior to mass spectrometry. In such embodiments HRT panel analytes maybe extracted using an TFLC extraction cartridge which captures theanalytes, then eluted and chromatographed on a second TFLC column oronto an HPLC or UPLC analytical column prior to ionization. Because thesteps involved in these chromatography procedures can be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature can result insavings of time and costs, and eliminate the opportunity for operatorerror.

It is believed that turbulent flow, such as that provided by TFLCcolumns and methods, may enhance the rate of mass transfer, improvingseparation characteristics. TFLC columns separate components by means ofhigh chromatographic flow rates through a packed column containing rigidparticles. By employing high flow rates (e.g., 3-5 mL/min), turbulentflow occurs in the column that causes nearly complete interactionbetween the stationary phase and the analyte(s) of interest. Anadvantage of using TFLC columns is that the macromolecular build-upassociated with biological fluid matrices is avoided since the highmolecular weight species are not retained under the turbulent flowconditions. TFLC methods that combine multiple separations in oneprocedure lessen the need for lengthy sample preparation and operate ata significantly greater speed. Such methods also achieve a separationperformance superior to laminar flow (HPLC) chromatography. TFLC oftenallows for direct injection of biological samples (plasma, urine, etc.).Direct injection is difficult to achieve in traditional forms ofchromatography because denatured proteins and other biological debrisquickly block the separation columns. TFLC also allows for very lowsample volume of less than 1 mL, preferably less than 0.5 mL, preferablyless than 0.2 mL, preferably about 0.1 mL.

Examples of TFLC applied to sample preparation prior to analysis by massspectrometry have been described elsewhere. See, e.g., Zimmer et al., J.Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367;5,919,368; 5,795,469; and 5,772,874. In certain embodiments of themethod, samples are subjected to protein precipitation as describedabove prior to loading on the TFLC column; in alternative preferredembodiments, the samples may be loaded directly onto the TFLC withoutbeing subjected to protein precipitation. Preferably, TFLC is used inconjunction with HPLC to extract and purify one or more HRT panelanalytes without subjecting the sample to protein precipitation. Inrelated preferred embodiments, purifying the sample prior to MS analysisinvolves (i) applying the sample to a TFLC extraction column, (ii)washing the TFLC extraction column under conditions whereby one or moreHRT panel analytes are retained by the column, (iii) eluting retainedHRT panel analytes from the TFLC extraction column, (iv) applying theretained material to an analytical column, and (v) eluting purified HRTpanel analytes from the analytical column. The TFLC extraction column ispreferably a large particle column. In various embodiments, one of moresteps of the methods may be performed in an on-line, automated fashion.For example, in one embodiment, steps (i)-(v) are performed in anon-line, automated fashion. In another, the steps of ionization anddetection are performed on-line following steps (i)-(v).

One means of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain LC techniques,including HPLC, rely on relatively slow, laminar flow technology.Traditional HPLC analysis relies on column packings in which laminarflow of the sample through the column is the basis for separation of theanalyte of interest from the sample. The skilled artisan will understandthat separation in such columns is a diffusional process and may selectHPLC instruments and columns that are suitable for use with HRT panelanalytes. The chromatographic column typically includes a medium (i.e.,a packing material) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlesinclude a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bondedsurface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18bonded alkyl groups, preferably C-18 bonded groups. The chromatographiccolumn includes an inlet port for receiving a sample directly orindirectly from a solid-phase extraction or TFLC column and an outletport for discharging an effluent that includes the fractionated sample.

In one embodiment, the sample is applied to the column at the inletport, eluted with a solvent or solvent mixture, and discharged at theoutlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted on a hydrophobic columnchromatographic system. In certain preferred embodiments, a C18analytical column is used (e.g., an XBridge C18 column from Waters,Corp. (3.5 μm particle size; 100×3.0 mm), a Hypersil Gold column fromThermoFisher (3 μm particle size; 100×3.0 mm), or equivalent). Incertain preferred embodiments, TFLC and HPLC are performed using HPLCGrade Ultra Pure water and 100% methanol as mobile phases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, TFLC may be used for purification of one or moreHRT panel analytes prior to mass spectrometry. In such embodiments, oneor more HRT panel analytes may be extracted using a TFLC extractioncolumn, then eluted and chromatographed on a second TFLC column or ontoan analytical HPLC column prior to ionization. For example, HRT panelanalyte extraction with an TFLC extraction column may be accomplishedwith a large particle size (50 μm) packed column. Sample eluted off ofthis column may then be transferred to an HPLC analytical column forfurther purification prior to mass spectrometry. In preferredembodiments, a large particle polymer based column, such as a Cyclone P®column from Cohesive Technologies, Inc. (50 μm particle size, 1.0×50mm), or equivalent is used as the TFLC column. Because the stepsinvolved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

Detection and Quantitation by Mass Spectrometry

In various embodiments, one or more HRT panel analytes may be ionized byany method known to the skilled artisan. Mass spectrometry is performedusing a mass spectrometer, which includes an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electronionization, chemical ionization, electrospray ionization (ESI), photonionization, atmospheric pressure chemical ionization (APCI),photoionization, atmospheric pressure photoionization (APPI), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. The skilled artisan will understand that the choice ofionization method may be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

The one or more HRT panel analytes may be ionized in positive ornegative mode to create one or more HRT panel ions. In preferredembodiments, the one or more HRT panel analytes are ionized byelectrospray ionization (ESI) in positive or negative mode; preferablynegative mode. In alternative preferred embodiments, the one or more HRTpanel analytes are ionized by atmospheric pressure chemical ionization(APCI) in positive or negative mode; preferably negative mode. Inrelated preferred embodiments, the one or more HRT panel ions are in agaseous state and the inert collision gas is argon or nitrogen.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, ions may be detected using a scanningmode, e.g., multiple reaction monitoring (MRM) or selected reactionmonitoring (SRM). Preferably, the mass-to-charge ratio is determinedusing a quadrupole analyzer. For example, in a “quadrupole” or“quadrupole ion trap” instrument, ions in an oscillating radio frequencyfield experience a force proportional to the DC potential appliedbetween electrodes, the amplitude of the RF signal, and the mass/chargeratio. The voltage and amplitude may be selected so that only ionshaving a particular mass/charge ratio travel the length of thequadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of one or moreHRT panel analytes. Methods of generating and using such standard curvesare well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, in preferredembodiments one or more isotopically labeled analogues of HRT panelanalytes (e.g., d₄-estrone (E1-d4), d₂-17α-estradiol (E2a-d2),d₅-17β-estradiol (E2b-d5), d₃-estriol (E3-d3), d₄-equilin (EQ-d4),d₅-17β-dihydroequilin (EQb-d5), d₃-equilenin (EN-d3),d₃-17β-dihydroequilenin (ENb-d3), d₄-estrone sulfate (E1s-d4),d₄-17β-estradiol sulfate (E2bs-d4), and d₂-Δ8,9-dehydroestrone (dE1-d2))may be used as internal standards. Numerous other methods for relatingthe amount of an ion to the amount of the original molecule will be wellknown to those of ordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, one or more HRT panel analytesare quantified in a sample using MS/MS as follows. The samples aresubjected to liquid chromatography, preferably TFLC followed by HPLC;the flow of liquid solvent from the chromatographic column enters theheated nebulizer interface of an MS/MS analyzer; and the solvent/analytemixture is converted to vapor in the heated tubing of the interface. TheHRT analytes contained in the nebulized solvent are then ionized. Theions, e.g. precursor ions, pass through the orifice of the instrumentand enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are massfilters, allowing selection of ions (i.e., selection of “precursor” and“fragment” ions in Q1 and Q3, respectively) based on their mass tocharge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ionsare fragmented. The first quadrupole of the mass spectrometer (Q1)selects for molecules with the mass to charge ratios of one of the HRTpanel analytes. Precursor ions with the correct mass/charge ratios areallowed to pass into the collision chamber (Q2), while unwanted ionswith any other mass/charge ratio collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral collision gas molecules and fragment. The fragment ionsgenerated are passed into quadrupole 3 (Q3), where the fragment ions ofthe selected HRT panel analyte are selected while other ions areeliminated. During analysis of a single sample injection, Q1 and/or Q3may be adjusted such that mass/charge ratios of one or more precursorion/fragment ion pairs specific to one HRT panel analyte are firstselected, followed at some later time by the selection of mass/chargeratios of one or more precursor ion/fragment ion pairs specific to asecond HRT panel analyte, optionally repeated at some later time for asmany HRT panel analytes as is desired. In particularly preferredembodiments, at least one precursor ion/fragment ion pair is selectedfor every HRT panel analyte in an analysis of a single sample injection,although the sequence of pair selection may occur in any order.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably negative ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of a HRT panel analytethat may be used for selection in quadrupole 3 (Q3). Preferred precursorion/fragment ions for HRT panel analytes and exemplary internalstandards are found in Table 1.

TABLE 1 Preferred Precursor Ion/Fragment Ion Mass to Charge Ratios ofHRT Panel Analytes and Exemplary Internal Standards Analyte Abbr. Parent(m/z) Fragment(s) (m/z) Equilenin EN 265.09 193.10, 221.0817α-Dihydroequilenin ENa 267.08 180.10, 195.13 17β-Dihydroequilenin ENb267.08 180.09, 195.12 Equilin EQ 267.06 115.07, 181.07Δ8,9-Dehydroestrone dE1 267.06 171.07, 195.08 d₃-Equilenin EN-d3 268.15182.08 17β-Dihydroequilin EQb 269.09 143.10, 193.10 Estrone E1 269.10143.09, 145.10 17α-Dihydroequilin EQa 269.10 169.10, 183.11d₃-17β-Dihydroequilenin ENb-d3 270.17 182.08 17α-Estradiol E2a 271.12143.1, 145.10 17β-Estradiol E2b 271.12 169.10, 183.10 d₄-Equilin EQ-d4271.16 243.14 d₄-Estrone E1-d4 273.13 147.08 d₂-17α-Estradiol E2a-d2273.20 147.08 d₅-17β-Dihydroequilin EQb-d5 274.15 213.10d₅-17β-Estradiol E2b-d5 276.13 187.08 Estriol E3 287.10 143.10, 171.10d₃-Estriol E3-d3 290.20 173.10 Estrone Sulfate E1s 349.01 143.08, 145.10d₄-Estrone Sulfate E1s-d4 352.9 147.09 Estradiol Sulfate E2s 351.02145.10, 183.10 d₄-17β-Estradiol Sulfate E2bs-d4 355.0 275.18d₂-Δ8,9-Dehydroestrone dE1-d2 269.02 209.05

In Table 1, above, a plurality of preferred fragment ions are listed forequilenin, 17α-dihydroequilenin, 17β-dihydroequilenin, equilin,Δ8,9-dehydroestrone, 17β-dihydroequilin, estrone, 17α-dihydroequilin,17α-estradiol, 17β-estradiol, estriol, estrone sulfate, and estradiolsulfate). In alternative embodiments, a single fragment ion may bedetected for these HRT panel analytes. For example, a single fragmention with a m/z of 181.1 may be detected for equilin; a single fragmention with a m/z of 171.07 may be detected for Δ8,9-dehydroestrone; asingle fragment ion with a m/z of 143.10 may be detected for17β-dihydroequilin; and a single fragment ion with a m/z of 169.10 maybe detected for 17α-dihydroequilin.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of each HRT panel analyte detected. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte using calibration standard curves basedon peaks of one or more ions of an internal molecular standard.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Sample Preparation

Blood was collected in a Vacutainer with no additives and allowed toclot 30 minutes at room temperature, 18° to 25° C. Serum was collectedfor further analysis. Samples that exhibited gross hemolysis and/orlipemia were excluded.

Example 2 Extraction of HRT Panel Analytes from Samples Using TFLC-HPLC

Serum was prepared for LC by pipetting 200 μL of patient serum into awell of a 96-well plate. 25 μL of a combined internal standard solutionwas added to each well along with 300 μL of 40% ethanol solution. Thesamples were incubated at room temperature for 30 to 45 minutes prior toLC.

Liquid chromatography was performed on some samples with a CohesiveTechnologies Aria TX-4 HTLC system using Aria OS V 1.5 or newersoftware. An autosampler wash solution was prepared using 30%acetonitrile, 30% methanol, 30% isopropanol, and 10% acetone (v/v).

In one example, the HTLC system automatically injected 75 μL of an aboveprepared sample into a Cyclone P® extraction column from CohesiveTechnologies, Inc. (50 μm particle size, 1.0×50 mm). The samples wereloaded at a high flow rate to create turbulence inside the extractioncolumn. This turbulence ensured optimized binding of HRT panel analytesto the large particles in the column and the passage of residual proteinand debris to waste.

Following loading, the flow direction was reversed and the sample elutedand transferred to either a XBridge C18 analytical column from Waters,Corp. (3.5 μm particle size; 150×3.0 mm) or a Hypersil Gold analyticalcolumn from ThermoFisher (3 μm particle size; 100×3.0 mm) in a columnoven/heater set at 40°. A binary HPLC gradient was applied to theanalytical column to separate HRT panel analytes from each other andother analytes contained in the sample. Mobile phase A was 0.1% aqueousammonium hydroxide and mobile phase B was 100% methanol. The approximateretention times of the various HRT panel analytes are shown in Table 2.

TABLE 2 Approximate Retention Times of HRT Panel Analytes Analyte Abbr.Approximate Retention Time Estrone Sulfate E1s 3.53 Estradiol SulfateE2s 3.53 Estriol E3 4.07 Equilenin EN 5.16 17β-Dihydroequilenin ENb 5.4817α-Dihydroequilenin ENa 5.88 17β-Dihydroequilin EQb 6.00Δ8,9-Dehydroestrone dE1 6.16 Equilin EQ 6.18 17α-Dihydroequilin EQa 6.2017β-Estradiol E2b 6.31 Estrone E1 6.44 17α-Estradiol E2a 6.62

These separated samples were then subjected to MS/MS for quantitation ofselected HRT panel analytes.

Example 3 Extraction of HRT Panel Analytes from Samples Using TFLC-UPLC

Processed serum samples were alternatively subject to TFLC-UPLC, ratherthan TFLC-HPLC extraction as described above. In this instance, the HTLCsystem automatically injected 75 μL of an above processed serum into aCyclone P® extraction column from Cohesive Technologies, Inc. (50 μmparticle size, 1.0×50 mm). The samples were loaded at a high flow rateto create turbulence inside the extraction column. This turbulenceensured optimized binding of HRT panel analytes to the large particlesin the column and the passage of residual protein and debris to waste.

Following loading, the flow direction was reversed and the sample elutedand transferred to either a Hypersil Gold analytical column fromThermoFisher (1.7 μm particle size; 50×2.1 mm) at 260 bar without heatat 0.45 mL/min, or an Acquity BEH C18 analytical column from Waters (1.7μm particle size; 50×2.1 mm) at 280 bar with heat set at 40°-50° at 0.5mL/min. A binary UPLC gradient was applied to the analytical column, toseparate HRT panel analytes from each other and other analytes containedin the sample. Mobile phase A was Ultra Pure Water (HPLC grade) andmobile phase B was 100% methanol.

These separated samples were then subjected to mass spectrometricanalysis for quantitation of selected HRT panel analytes.

Example 4 Quantitation of HRT Panel Analytes by MS/MS

MS (and MS/MS) was performed using a Thermo TSQ Quantum Ultra MS/MSsystem. The following software programs all from ThermoElectron wereused in the Examples described herein: Tune Master V 1.5 or newer,Xcalibur V 2.0.7 SR1 or newer, TSQ Quantum 1.5 or newer, LCQuan V 2.5.6or newer, and XReport 1.0 or newer. The solvent/analyte mixture wasconverted to vapor in the heated tubing of the interface. Analytes inthe nebulized solvent were ionized by heated ESI.

First, a Total Ion Count was measured for a single sample injectioncontaining thirteen HRT panel analytes. This scan is shown in FIG. 1 anddemonstrates the total ion count versus time.

Then MS/MS analysis was conducted by passing ions to the firstquadrupole (Q1), which selected ions with a desired parent mass tocharge ratio. Ions entering quadrupole 2 (Q2) collided with argon gas togenerate ion fragments, which were passed to quadrupole 3 (Q3) forfurther selection. Simultaneously, the same process using isotopedilution mass spectrometry was carried out with selected isotope-labeledinternal standards. All of the selected masses for each HRT panelanalyte and selected isotope-labeled internal standards are listed inTable 1, above.

FIG. 2 shows the results of monitoring the mass transitions listed inTable 1 for HRT panel analytes in a single sample injection. As seen inthis Figure, all 13 analytes were detected in a single sample injection.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount of17β-estradiol and estrone in an individual undergoing hormonereplacement therapy (HRT), the method comprising: a. ionizing a samplecomprising the body fluid of an individual undergoing HRT underconditions suitable to produce one or more ions of 17β-estradiol andestrone detectable by mass spectrometry; b. determining the amounts ofone or more ions of 17β-estradiol and estrone by tandem massspectrometry; and c. using the amounts of one or more ions from each of17β-estradiol and estrone to determine the amounts of each of17β-estradiol and estrone in the body fluid sample of the individual. 2.The method of claim 1, further comprising calculating a ratio of thecombined levels of sulfated 17β-estradiol and estrone compared to thecombined levels of non-sulfated 17β-estradiol and estrone, wherein ifthe non-sulfated 17β-estradiol and estrone are predominant, a decreasein HRT dosage is indicated.
 3. The method of claim 1, further comprisingcalculating a ratio of the combined levels of 17β-estradiol and estronecompared to the combined levels of non-sulfated 17β-estradiol andestrone, wherein if the sulfated estrogenic 17β-estradiol and estroneare predominant, an increase in HRT dosage is indicated.
 4. The methodof claim 1, further comprising determining the amount of one or more ofestrone sulfate (E1s), 17α-estradiol (E2a), estradiol sulfate (E2s),estriol (E3), equilin (EQ), 17α-dihydroequilin (EQa), 17β-dihydroequilin(EQb), equilenin (EN), 17α-dihydroequilenin (ENa), 17β-dihydroequilenin(ENb), or Δ8,9-dehydroestrone (dE1).
 5. The method of claim 1, whereinthe sample has been purified by liquid chromatography prior toionization.
 6. The method of claim 5, wherein liquid chromatography isselected from the group consisting of high performance liquidchromatography, ultra high performance liquid chromatography, andturbulent flow liquid chromatography.
 7. The method of claim 1, whereinthe sample has been purified by turbulent flow liquid chromatography andeither high performance liquid chromatography or ultra high performanceliquid chromatography prior to ionization.
 8. The method of claim 1,wherein the sample comprises serum or plasma.
 9. The method of claim 1,further comprising determining the amount of one or more internalstandards by tandem mass spectrometry.
 10. The method of claim 9,wherein each internal standard comprises an isotopically labeled17β-estradiol and estrone.
 11. The method of claim 10, wherein theinternal standard is d₄-estrone (E1-d4) and d₅-17β-estradiol (E2b-d5).12. The method of claim 1, wherein said ionizing comprises ionization byatmospheric pressure chemical ionization (APCI).
 13. The method of claim1, wherein said ionizing comprises ionization by electrospray ionization(ESI).